1. Field of the Invention
As the use of illicit drugs in this country has increased, public concern over the problems associated with its effects has grown into a major concern. This concern has led to workplace drug testing in order to identify, treat, and remove active drug users from the workforce. This trend started in the military, and spread rapidly to law enforcement and any xe2x80x9csafety-sensitivexe2x80x9d private sector jobs such as airline pilots, truck drivers, and active crew members of public transportation. These initial strides into drug testing in the workplace revealed the obtrusive incursion of drug use and abuse in the daily lives of a significant portion of Americans. Further research indicated the staggering costs to public and private industry in terms of lost productivity, increased health care costs, and human suffering and death due to this scourge of drug abuse. As a result, drug testing has rapidly spread to all areas of the public and private sector. The vast majority of workplace drug testing has taken the form of urine testing, because of ease of collection, low cost, and effective indication of recent drug use. Other forms of testing include analysis of blood, saliva, sweat, and hair.
Gamma-hydroxybutyrate (4-hydroxybutyrate, 4-hydroxybutyric acid, Gamma-hydroxybutyric acid, 4-hydroxybutyric acid sodium salt, GHB) was first used for anesthetic purposes in 1961, because it was unpredictable and caused adverse effects, its use was discontinued. Later, GHB was used by body builders for muscle building and weight control. Presently, the U.S. DEA (Drug Enforcement Agency) is investigating GHB to see if it should be a controlled substance. The U.S. FDA (Food and Drug Administration) list GHB as an unapproved drug except for investigational use in the treatment of narcolepsy. Common names for GHB are Scoop, Georgia Home Boy, Grievous Bodily Harm, Liquid Ecstasy, and Cherry Meth among others. Its precursor GBL (gamma-butyrolactone) is used as a GHB substitute and once ingested rapidly converts to GHB. The pharmacological effects of both GHB and GBL are similar and the range of analgesic effect (euphoria) are similar.
In the 1990""s GHB has become a popular drug of abuse on college campuses, bars, and dance clubs and is called the xe2x80x9cdate rapexe2x80x9d drug. The abuse of GHB has enormous sociological and economic impact on our society. A typical xe2x80x9cdate rapexe2x80x9d scenario is as follows: The victim(s), usually women, are in a bar, they drink a beverage that has been laced with GHB by a rapist, the victim then becomes catatonic and is usually agreeable with anyone and everyone around them. They can become unconscious and then of course are susceptible to the rapist desires. Later, the victim(s) wakes up completely disoriented, naked and robbed. This type of horrific tragedy is occurring on a daily basis. The victims of xe2x80x9cdate rapexe2x80x9d are not only exposed to the physical assault of rape and robbery, but to the contraction of diseases such as AIDS and STD""s. The damage caused and the consequences of such occurrences are immeasurable.
Accordingly, a need exists for providing an easy and convenient manner by which to make a determination of the presence of GHB in urine, in a beverage, or other biological fluids or liquids. A further need exists for a convenient manner by which such determinations may be made by using rapid analysis manual techniques (such as a dipstick or lateral flow devices) and automated techniques that will advance the art significantly. And, the most important need is for a device that would detect GHB using just a single assay that does not require an extraction process or lactone conversion. This would be a marked advancement in the art and would result in the savings of millions of dollars to the drug testing laboratories required to perform GC (gas chromatography) or GCMS (gas chromatography mass spectrometry) testing for GHB and obviously this savings would be passed on to the end user (the businesses which initially request drug""s of abuse assays on perspective and current employees). To explain further, the drug testing laboratory would normally perform GC (gas chromatography) or GCMS (gas chromatography mass spectrometry) assay for GHB. The necessary time to perform these assays is burdensome to the laboratory through cost for tech time, reagents, and turnaround time to mention a few. The alternative to this would be the significant advancement in the art that the present device offers which is the capability to detect GHB without lengthy extraction processes of the current art with a single assay.
2. Description of the Related Art
This invention is in the field of toxicology. More specifically, this invention provides test strips (i.e. dry chemistry dipsticks, or on-site test modules utilizing thin layer chromatography in a lateral flow format, or other similar technology to the test strip) and liquid chemistry reagents for use in the detection of GHB with a single assay in aqueous fluids to include but not limited to urine, saliva, serum, blood, sweat extracts, liquid homogenates of hair and liquids such as beverages, soft drinks, mixed drinks to included alcohol, etc.
It is known that the polarity and small molecular size of the GHB molecule and the lack of detectability of the GHB by UV (ultra violet), chromatographic, and spectrophotometric means complicates detection. GHB is relatively unstable and will form the GHB lactone derivative when heated and under acidic conditions. GHB can cause euphoria at less than 50 ug/mL to marked central depression, sleep, coma and death. Currently, all of the methods to detect GHB use and/or solid-phase extraction, liquid-liquid extraction, silyl-derivatization, then GCMS. These methods mentioned are very time consuming, expensive (costing the laboratories, companies ordering drug testing, and general public millions of dollars per year), and labor intensive. The GC-MS, assay is typically performed to verify the urines that screen positive for drugs of abuse. The GC-MS analysis costs 100 times as much as the initial screen ($100 vs $1). Every additional unnecessary GC-MS performed drives up the overall cost of drug testing.
The novel invention described herein describes a method to determine the presence or absence of GHB and its precursor(s) in urine or other fluids by liquid and dry chemistry test means which has not been taught prior to the present art. It should be noted that GHB is not normally found in urine.
There are no published, taught, or even mentioned methods of the present arts technology to detect GHB or GBL in urine by the present arts techniques.
Again, a thorough search of patents and research revealed no relative art (i.e., prior art) with any correlation to this technology. The art of testing for GHB or GBL in urine or other fluids as previously delineated in the literature describe various techniques including methods for solid-phase extraction, liquid-liquid extraction, silyl-derivatization, then GCMS. No reference, however, has described this new art as delineated here. The previous art will be enumerated here to further illustrate the unique advancement in the field of GHB and GBL detection. It has been acknowledged in the art that random urinary sample matrices are very complex, and consist of many urinary constituents which create strong buffering and interference problems (e.g. cannibal like enzymes such as protease). In addition, disease states will significantly impact the nature of urinary contents. Urine is also the repository of all of the body""s waste products including excess parent nutrients, vitamins, drugs, and their metabolites. These waste chemicals vary from person to person and significantly contribute to the individual uniqueness that makes assay design for urinary constituents more difficult than any other body fluid. All of these factors impact an assay""s ability to obtain acceptable precision and accuracy. The ability of an assay to analyze a biological liquid such as saliva, therefore, rarely ever translates to an effective assay for urine. Therefore the present invention""s ability to effectively cope with random urine samples and biological fluids or other fluids such as a beverage(s) makes it unique.
Patent, U.S. Pat. No. 3,603,957, discloses the use of assay test strips, but fails to teach a method for the determination of GHB or GBL of a test sample submitted for drugs of abuse testing. It also doesn""t teach a method to determine the presence or absence of any substance such as GHB or GBL. The patent doesn""t teach the use of the present art""s reaction formula to dry chemistry format called a dipstick or lateral flow technology that not only is completely novel, but prevents cross contamination between test pads typically found on a test strip (dipstick). In addition, this patent also failed to mention any methods for determination of GHB or GBL by dipstick, lateral flow, calorimetric, liquid reagent (automated) or other suitable means.
Another patent, U.S. Pat. No. 4,301,115, discloses the use of assay test strips, and the ability of the assay strips to resist cross contamination between reactant areas (chemically impregnated test pads), but fails to teach a method for the determination of GHB or GBL in a sample of fluid. The patent doesn""t teach the use of the dry chemistry format utilizing either a dipstick or lateral flow device, liquid reagent (automated) method or mention any methods for determination of GHB or GBL.
Another patent, U.S. Pat. No. 5,447,837, does mention the use of assay test strips but again fails to disclose a method for the determination of GHB or GBL. This is a method for detection of an antigenic substance in human, biological samples. This patent also fails to mention the use of a reaction formula that is adaptable to the dry chemistry format utilizing either a dipstick or lateral flow device. It also doesn""t teach a method to determine the presence or absence of any substance such as GHB or GBL. In addition, this patent also failed to mention any methods for determination of GHB or GBL by dry chemistry, liquid chemistry, calorimetric, or other suitable means.
Published literature and the prior art describes techniques such as ELISA that have been used to determine the presence of drugs of abuse, but these technologies have no relevant bearing on the present device. Previously taught technologies include measurement of GHB or GBL using GC or GCMS techniques and the required extraction procedures inherent (required) prior to analysis by GC or GCMS.
Therefore, in a nutshell, the present device provides an absolute novel approach to GHB or GBL testing and lateral flow testing using dry chemistry test pads and automated liquid reagent testing.
Not surprisingly, it is known and is illustrated here that a great need exists in the field of drug testing for rapid, economical, and effective method for the detection of GHB or GBL on samples submitted for testing, whether liquid chemistry and/or dry chemistry methodology using dipsticks or lateral flow test devices (for single use and for on-site collections). The present invention does detect GHB or GBL effectively with a single assay and therefore and accordingly, the present device provides an easy and convenient manner by which to make a determination of the presence or absence of GHB or GBL in a fluid. The present art""s use of lateral flow also enables the removal of any interference of any cross over of reagents or fluid from one test pad to another which is one of the exclusive problems with dipsticks.
It is clear that a need exists for a convenient manner by which a determination of GHB or GBL can be made utilizing a rapid automated analysis utilizing a liquid reagent format of the present device or manual analysis in the form of dry chemistry (dipstick) and/or lateral flow test devices. These and other advances in the current state of the art will become evident in view of the present specification and claims.
Briefly stated, the present invention relates to the test devices for detecting the presence of GHB and or its precursor GBL (gamma-butyrolactone) in a liquid test sample and the methods for making said devices. GBL is also used as a GHB substitute. Once ingested, GHL is rapidly converted to GHB. GHB can also be converted to GBL by acid catalysis. This invention is in the field of drug testing. More specifically, this invention provides dry chemistry test strips (i.e. dipsticks, or dry chemistry and lateral flow [thin layer chromatography] test means) or automated or manual liquid reagent means for use in the detection of GHB or GBL in biological samples (e.g. urine, blood, serum, saliva, sweat extracts, and hair homogenates) or other fluids such as beverages, water, soft drinks, alcoholic drinks to name a few. This invention achieves this goal by measuring the presence of GHB or GBL in a test sample. And, this invention provides a unique method for preventing cross contamination between test pads (reactant areas) on dipsticks by the present inventions use of the dipstick test pad and lateral flow device technology. This invention provides a previously unavailable dry chemistry or liquid chemistry method for determining GHB or GBL presence in a test sample by measuring the presence of GHB or GBL.
The present invention encompasses a method that can utilize several different techniques. The techniques would employ the manual method using dry chemistry dipsticks and the method of combining dry chemistry dipstick reactant areas (test pads) with lateral flow thin layer chromatography or the method of using a liquid reagent that is compatible with automated analyzers that provide high speed quantitative analysis which would be much less labor intensive than the manual methods providing a savings in time and money. The widespread utility of the present art also provides the drug testing laboratory, over-the-counter user, individual, police agency, drug testing collection site (where the urine is actually collected), or other users the choice of using the dry chemistry (manual) or automated liquid means which ever method best suits their situation or needs.
The present arts technique utilizes two dry chemistry techniques, one is dipsticks, which is a carrier dependent, rapid test that uses absorbent medium such as paper which have been impregnated with a chemical formulation to detect adulteration. After dipping one (dipstick) into a liquid test sample, a reaction takes place. Said resulting reaction will yield a color change indicating a positive or negative result (i.e. presence or absence of GHB or GBL). The other technique is the use of lateral flow in combination with a dry chemical test pad. The lateral flow device is a rapid test that uses absorbent medium such as paper which has been impregnated with the chemical formulation to detect GHB or GBL. The paper, after impregnation, is then placed on a lateral flow medium, such as nitrocellulose paper, glass fiber paper, or other suitable wicking material that will deliver the test sample to the impregnated paper. The lateral flow device works by dipping one end of the lateral flow device (LFD) into a sample (urine for example). The urine migrates up (along) the paper (or absorbent material) to the reactive sites (test paper) containing reagents (reactive ingredients). The urine constituents react with the assay reagents during the migration process and yield visible results. The urine can also be droppered onto the LFD and the sample will then migrate along the paper.
The ease of use and rapid results obtained by the present art""s methodology illustrate the unique utility of this testing technique. In addition, very little technical expertise is required to perform this type of assay (no instrumentation required). Furthermore, the early detection of GHB or GBL facilitates the prevention of the xe2x80x9cdate rapexe2x80x9d syndrome. This novel concept for GHB or GBL monitoring provides an enormous savings of time and money. The present arts method(s) of GHB or GBL testing utilizing these techniques are currently not available and have never been taught.
An important aspect of GHB or GBL testing in fluids is the sensitivity of the test method. Both techniques taught here have an effective sensitivity range comparable to the GC-MS target range. The sensitivity can also be adjusted to indicate a large amount present as would be the case if the sample tested were a soft drink spiked with GHB or GBL. Obviously, the amount in a sample directly spiked with GHB or GBL would be much higher in concentration than the amounts found in urine. The dipstick test and LFD dipstick hybrid (to be known as the LFD hybrid) have a quantitative to qualitative assay range. The results are evaluated via one of following categories: negative, positive, or quantitatively.
The present arts technique also utilizes a liquid chemistry test means that allows for rapid analysis via an automated analyzer that can yield high speed quantitative results. This will result in rapid test results, improved accuracy, lowered labor cost, and better turn around on a high volume of test. This automated method is only limited by the speed of the automated analyzer. Some analyzers currently on the market are capable of over 10,000 test per a hour. The ability of the present art to perform a single assay on a high speed automated analyzer that is capable of detecting GHB or GBL has never been present or taught in the prior art.
It is currently known in the art that enzyme and antigen/antibody reaction kinetics are related to the rate of change in analytical, biological systems. The variables that affect this rate of change include concentration of reactants and product, temperature, pH, ionic strength, buffer strength, and other parameters. The present art""s innate and unique ability enables it to determine the presence of GHB or GBL in fluids. As it is known in the art urine is a very complex matrix and the measurement of GHB or GBL in fluids such as urine has to take into account many factors which will affect the assay.
The composition of the formulation to be applied to the dry chemistry dipstick, LFD hybrid and liquid chemistry method are composed of indicator(s) (visible calorimetric), and buffer(s).
Briefly stated, the present invention relates to test devices for measurement of GHB or GBL in urine but could also work in other biological matrices such as blood, saliva, hair or other fluids and the procedures for making said test means. This invention is in the field of clinical diagnostics. More specifically, this invention provides dry chemistry dipsticks (DCD""s or on-site test modules), thin layer lateral flow chromatographic dry chemistry technology (LFD""s), and the combination of both in a unique hybrid that is not known prior to the present art and liquid chemistry reagents for automated and manual use. That is to say (in it""s simplest terms) that this unique hybrid (LFD) will encompass the use of a dry chemistry test pad resting on the surface of a wicking material (such as nitrocellulose) acting as a fluid delivery device. This new art can utilize aqueous, biological specimens including urine, saliva, sweat extracts, blood, serum and other fluids (such as water or soft drinks, etc.). Thus, this invention provides a unique method for GHB or GBL measurement utilizing rapid test devices including the automated method as well as the DCD, and LFD methodology thereby enabling in-home, workplace, and recreational testing through over-the-counter (OTC) sales. This is an enormous advancement in the art. These advances and improvements of the present device over the prior art provides the public safety, health care and drug testing industry with powerful new clinical and diagnostic tools.
A thorough search of the literature reveals no relative art resembling this technology; therefore, this invention is clearly a novel creation, and is not obvious to anyone skilled in the art of toxicology and clinical chemistry.
The instant invention is a single assay in the form of a liquid chemistry reagent, dry chemistry dipstick or lateral flow device in conjunction with using a dry chemistry test pad for the detection of GHB or GBL in sample matrices consisting of urine and other biological specimens (e.g. saliva, serum, blood, sweat extracts, and hair homogenates) or fluids such as beverages such water, soft drinks, beer, or mixed drinks. The GHB or GBL detection assay that makes up the instant invention may take the form of dry chemistry dipsticks or dry chemistry test pad lateral flow hybrid, both of which are composed of some or all of the following compounds: buffer(s) and color indicator(s), hereinafter referred to as the adulteration reagent or the liquid automated reagent designed for high speed automated analyzers also composed of some or all of the following compounds: buffer(s) and color indicator(s). It can be noted that the liquid reagent method could also be used manually employing spectrophotometers or other types of visual detection technology. Buffering of the reactants is critical to the GHB or GBL reagent, because pH plays a vital role in the reaction kinetics. In the case of the dipstick (or dry chemistry dipstick, DCD) and the dipstick/lateral flow hybrid (which can be known as the xe2x80x9cDLFHxe2x80x9d device), GHB or GBL reagent components are impregnated on the test strip pad composed of solid, absorbent carrier(s), usually known as test pads. In the case of dipsticks, these test pads are typically affixed to a solid support (usually plastic). This device is then submerged in the liquid test sample, removed, and a measurable (i.e. visible) response is observed. Or in the case of the DLFH, the dry chemistry test pad is chemically impregnated identically to the dipstick. The test pad is then placed in fluid (direct) contact with lateral flow paper (such as nitrocellulose). This device is then exposed to a fluid (urine for example). The urine then migrates to the location of the test pad, saturates the test pad, and the reaction takes place.
The GHB or GBL reagents of the device constitute the heart of the analytical response provided by it, and is comprised of one or more reagent compositions responsive to any number of chemical components made up of GHB or GBL or are analogs or precursors of GHB. The reagents, in the broadest sense produces a detectable manifestation of the presence of GHB or GBL; the detectable manifestation can be a measurable response in the form of the appearance or disappearance of a color, or the changing of one color to another. Said measurable response may also be evidenced by a change in the amount of light reflected or absorbed during the reaction of interest. The analytical arts are replete with examples of these types of detectable responses.
In the present invention, there is provided a dry chemistry test strip in the form of a dipstick or DLFH for the detection of GHB or GBL in urine (or other biological fluids including saliva, sweat extracts, serum, blood, and hair homogenates) and fluids such as beverages to included but not limited to water, soft drinks, beer, or mixed drinks (possibly containing alcohol) comprising a solid, carrier matrix in the form of a dry chemistry dipstick containing an indicator compound and buffer.
The present technology does not predict or forecast the obvious advancement in the art to encompass the present invention, nor does it hint at the extraordinary improvement the present invention provides in the field of GHB or GBL detection. While urine is the sample matrix of choice for this instant invention (and for the immunoassays currently in general use for drug abuse screening) it is well within the scope of this novel invention to utilize it in the analysis of other sample matrices including saliva, sweat extracts, serum, hair homogenates, gastric contents, cerebral spinal fluid, blood and fluids such as beverages to included but not limited to water, soft drinks, beer, or mixed drinks (possibly containing alcohol).
The remarkable discovery of the new art formula will require the presence of an indicator(s) for GHB or GBL in urine (as well as the other matrices mentioned) that was unknown prior to this art. The newly discovered and suitable indicators and compounds that are reactive to the presence of GHB or GBL are but, not limited to the following; hydroxybutyrate dehydrogenase, esterase, 3-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate dehydrogenase, carboxyl esterase, carboxylic-ester hydrolase, xcex2-hydroxybutyrate dehydrogenase, [R]-3-hydroxybutanoate, NAD (nicotinamide adenine dinucleotide)+ oxidoreductase, NAD (xcex1-nicotinamide adenine dinucleotide) or analogs of NAD such as 3-acetylpyridine adenine dinucloetide, 3-acetylpyridine hypoxanthine dinucleotide, xcex2-nicotinamide adenine dinucleotide-agarose, nicotinamide 1,N6-ethenoadenine dinucleotide, nicotinamide guanine dinucleotide, nicotinamide hypoxanthine dinucleotide, nicotinic acid adenine dinucleotide, 3-pyridinealdehyde adenine dinucleotide, thionicotinamide adenine dinucleotide and xcex1-hydroxybutyrate dehydrogenase, NADP (xcex1-nicotinamide adenine dinucleotide phosphate) or analogs of NADP such as xcex2-nicotinamide adenine dinucleotide phosphate, 3-acetylpyridine adenine phosphate, xcex2-nicotinamide adenine dinucleotide 2xe2x80x2,3xe2x80x2-Cyclic monophosphate, xcex2-nicotinamide adenine dinucleotide 3xe2x80x2-phosphate, nicotinamide 1,N6-etheno adenine dinucleotide phosphate, nicotinamide hypoxanthine dinucleotide phosphate, thionicotinamide adenine dinucleotide phosphate and anti-gamma-hydroxybutyrate, gamma-butyrolactone dehydrogenase, anti-gamma-butyrolactone, alpha-nicotinamide adenine dinucleotide phosphate, beta-nicotinamide adenine dinucleotide phosphate and all analogs of the afore mentioned, and can be selected from the following group consisting of NBT (nitro blue tetrazolium), phenazine methosulfate, tetranitroblue tetrazolium, napthol AS-TR phosphate, methylene blue, Fast red, napthol-AS-MX, napthol AS-TR phosphate, thymol blue, bromcresol green, methyl red, cresol red, metanil yellow, m-cresol purple, xylenol blue, thymol blue, tropeolin OO, quinaldine red, xcex1-dinitrophenol, methyl yellow; dimethyl yellow, bromophenol blue, tetrabromophenol blue, bromochlorophenol blue, Congo red, methyl orange, p-ethoxychrysoidine hydrochloride, napthyl red, alizarin sodium sulfonate, bromocresol green, xcex3-dinitrophenol, methyl red, lacmoid, chlorophenol red, benzoyl auramine G, bromocresol purple, bromophenol red, p-nitrophenol, bromthymol blue, phenol red, p-quinonemono(bis-4-oxyphenylmethide), neutral red, quinoline blue, xcex1-naphtholphthalein, tropeolin OOO; xcex1-napthol orange, ethyl bis(2,4-dinitrophenyl)acetate, di-o-cresolphthalide, phenolphthalein, thymolphthalein, dimethylphenolphthalein, alizarin yellow GG; salicyl yellow, alizarin yellow R, Nile blue, 2,4,6-trinitrophenylmethyl-nitramine, tropeolin O, triphenylrosaniline sulfonic acid (sodium or potassium salt), indigo carmine, nitrobenzene, bromcresol green, bromcresol purple, bromchlorophenol blue, brilliant yellow, brilliant blue R, brilliant cresyl blue ALD, brilliant blue G, brlliant black BN, bromthymol blue, bromphenol red, bromphenol red, bromoxylenol blue, coomasie blue, azolitmin, litmus, pyrogallosulfonphthalein, pyrogallo red-molybdate, alcohol dehydrogenase, ABTS (2,2xe2x80x2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)), 4-aminoantipyrine (4AAP), tetramethylbenzidine (TMB), o-phenylenediamine (OPD), o-dianisidine, 5-aminosalicylic acid (5AS), 3,3xe2x80x2-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), 4-chloro-1-napthol (4C1N), AEC (3-Amino-9-ethyl carbazole), dimethyl-2,5-dihydroperoxyhexane, Bis{4-[N-(3xe2x80x2-sulfo-n-propyl)-N-n-ethyl]amino-2,6-dimethylphenyl}methane (Bis-MAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS), N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS), N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-(3-sulfopropyl)aniline (HALPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-aniline (DAOS), N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS), N-Ethyl-N-(3-sulfopropyl)aniline (ALPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylani line (MAO), N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB), pyrogallol, 2,4-Dichlorophenol, N,N-Diethyl-m-toluidine, p-Hydroxybenzene Sulfonate, N,N-Dimethylaniline, 3,5-Dichloro-2-Hydroxybenzenesulfonate, 2,4,6-tribromo-3-hydroxybenzoic acid, Sodium N-Ethyl-N-(3-Sulfopropyl)-m-Anisidine, hydroxybenzoic acid, 4-hydroxybenzoic acid, N-Ethyl-N-(2-hydroxy-3-Sulfopropyl)-m-toluidine, AEC (3-Amino-9-ethyl carbazole), 2-5, dimethyl-2,5-dihydroperoxyhexane, Bis{4-[N-(3xe2x80x2-sulfo-n-propyl)-N-n-ethyl]lamino-2,6-dimethylphenyl}methane (Bis-MAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS), N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS), N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-(3-sulfopropyl)aniline (HALPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-aniline (DAOS), N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS), N-Ethyl-N-(3-sulfopropyl)aniline (ALPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO), N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB), 3-Methyl-2-benzothiazolinonehydrazone, Dimethylaniline, 5-bromo-6-chloro-3-indoxyl-beta-D-galacatopyranoside, 4-Aminophenyl-beta-D-galactopyranoside, 3-indoxyl-beta-D-galactopyranoside (blue), 5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside (blue), 5-Bromo-3-indoxyl-beta-D-galactopyranoside (blue), 6-chloro-3-indoxyl-beta-D-galactopyranoside (salmon), 6-Fluoro-3-indoxyl-beta-D-galactopyranoside, 8-Hydroxyquinoline-beta-D-galactopyrano-side, 5-Iodo-3-indoxyl-beta-D-galactopyranoside (purple), N-Methylindoxyl-beta-D-galactopyranoside, 2-Nitrophenyl-beta-D-galactopyranoside, 4-Nitrophenyl-beta-D-galactopyranoside, Naphthol AS-BI-beta-D-galactopyranoside, 2-Naphthyl-beta-D-galactopyranoside (yellow), 4-Methylumbelliferyl-beta-D-glucuronic acid, beta-D-Galactosidase, Iodo-3-indoxyl-beta-D-galactopyranoside, alpha-L-Galactosidase, Iodo-3-indoxyl-alpha-L-beta-Galactosidase, glycosidase, beta-Cellobiosidase, cellobioside, beta-D-Cellobiosidase, 5-Bromo-4-chloro-3-indoxyl-beta-D-cellobioside, 5-Bromo-6-chloro-3-indoxyl-beta-D-cellobioside, 4-Nitrophenyl-beta-D-cellobioside, 1-Naphthyl-cellobioside, 4-Methylumbelliferyl-beta-D-cellobioside, Arabinosidase, Fucosidase, Galactosaminidase, Glucosaminidase, Glucosidase, Glucuronidase, Lactosidase, Maltosidase, Mannosidase, and Xylosidase. Their corresponding substrates, Arabinopyranoside, Fucopyranoside, Galactosaminide, Glucosaminide, Glucopyranoside, Glucuronic acid, Lactopyranoside, Maltopyranoside, Mannopyranoside, Xylopyranoside, 5-Bromo-4-chloro-3-indoxyl, 5-Bromo-6-chloro-3-indoxyl, 6-chloro-3-indoxyl, 5-Bromo-3-indoxyl, 5-Iodo-3-indoxyl, 3-indoxyl, 2-(6-Bromonaphthyl), 6-Fluoro-3-indoxyl 2-Nitrophenyl, 4-Nitrophenyl, 1-Naphthyl, Naphthyl AS-BI, 2-Nitrophenyl-N-acetyl, 4-Nitrophenyl-N-acetyl, 4-Methylumbelliferyl, glycosidase enzyme, carboxyl esterase, cholesterol esterase, sulfatases (e.g. Aryl sufatase), phosphatases (e.g. Alkaline phosphatase), carboxyl esterase, 6-chloro-3-indoxyl butyrate, aryl sulfatase, 5-bromo-4-chloro-3-indoxyl sulfate, alkaline phosphatase, and 2-naphthyl phosphate. It is understood that the present arts discovery of the use of indicators such as the ones mentioned above or others that have not been mentioned that are sensitive to GHB or GBL biological matrices such as urine and other fluids are capable of producing a detectable response in the presence of GHB or GBL are within the present art. Therefore the use of GHB or GBL indicators that are not mentioned here would fall within the spirit and scope of the present invention. These indicators form indicatorcomplexes with GHB or GBL for determining the presence or amount of gamma-hydroxybutyrate or gamma-butyrolactone in a sample, said method(s) comprising contacting said sample with an indicator which specifically binds to gamma-hydroxybutyrate or gamma-butyrolactone to form an indicatorcomplex; and, measuring said indicatorcomplex to determine the presence or amount of said gamma-hydroxybutyrate or gamma-butrylactone in said sample.
This new art formula will require appropriate buffering. Suitable buffers may include any of the following (referred to here by their commonly used acronyms): citrate, borate, borax, sodium tetraborate decahydrate, sodium perchlorate, sodium chlorate, sodium carbonate, TRIS (Tris[hydroxymethyl]aminomethane), MES (2-[N-Morpholino]ethanesulfonic acid), BIS-TRIS (bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane; 2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA (N-[2-Acetamidol]-2-iminodiacetic acid; N-[Carbaoylmethyl]iminodiacetc acid), ACES (2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid; N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES (PiperazineN-Nxe2x80x2-bis[2-ethanesulfonic acid)]; 1,4-Piperzinedethanesulfoic acid), MOPSO (3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRIS PROPANE (1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES (N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid; 2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS (3-[N-Morpholino]propanesulfonic acid), TES (N-tris[Hydroxymethyl]methyl-2-aminomethanesulfonic acid; 2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid), HEPES (N-[2-Hydroxyethyl]piperazine-Nxe2x80x2-[2-ethanesulfonic acid]), DIPSO (3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), TAPSO (3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesulfonic acid), HEPPSO (N-[2-Hydroxythyl]piperazine-Nxe2x80x2-[2Hydroxypropanesulfonic acid]), POPSO (Piperazine-N,Nxe2x80x2-bis[2-hydroxypropanesulfonic acid]), EPPS (N-[2-Hydroxyethyl]piperazine-Nxe2x80x2-[3-propanesulfonic acid), TEA (triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine; N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE (N,N-bis[2-Hydroxyethyl]glycine), TAPS (N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid; ([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]arnino)-1-propanesulfonic acid), AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), CHES (2-[N-Cyclohexylamino]ethanesulfonic acid), CAPSO (3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP 2-Amino-2-ethyl-1-propanol, CAPS (3-[cyclohexylamino]-1-propanesulfdnic acid), hydrochloric acid, phosphoric acid, lactic acid, sulfuric acid, nitric acid, chromic acid, boric acid, perchloric acid, potassium hydrogen tartrate, potassium hydrogen phthalate, calcium hydroxide, phosphate, bicarbonate, sodium hydroxide, potassium hydroxide, oxalate or succinate. Other buffers with an effective pK and pH range, and capacity suitable for maintaining the sample-reagent mixture within the required parameters of the assay""s reaction mechanism may be added to the above group.
Manufacture of the dry chemistry dipsticks may require the addition of thickeners as taught in the art. Some compounds commonly used for this purpose include: polyvinylpyrrolidone, algin, carragenin, casein, albumin, methyl cellulose, and gelatin. The typical range of concentration for these thickeners is about 0.5 to 5.0 g. per 100 ml. Wetting agents or surfactants are also typically used in dry chemistry. For dry chemistry applications, wetting agents aid in even distribution of the chemicals and promote even color development. Acceptable wetting agents can be hydrophilic polymers, or cationic, anionic, amphoteric, or nonionic species. Some commonly used wetting agents include sodium dodecyl-benzene sulphonate, sodium lauryl sulphate, benzalkonium chloride, N-lauroylsarcosine sodium salt, Brij-35, Tween 20, Triton X-100, dioctyl sodium sulphosuccinate, and polyethylene glycol 6000. Wetting agents can be added to dipstick impregnation solutions in amounts of 0.5% to 5.0%, and 0.1% to 1.0% in liquid reagents.
Color enhancers may be used such as sucrose, lactose, glucose or other compounds. Color enhancement can be defined as intensification and/or alteration in some manner the color that is produced by the reaction to improve the measurement of the detectable response.
The production of dry chemistry test strips for the present invention can utilize any form of absorbent, solid phase carrier including filter paper, cellulose or synthetic resin fleeces in conjunction with liquid solutions of reagent compositions in volatile solvents. This can be carried out in one or more impregnation steps. Each impregnation may contain one or more of the chemical compounds making up the assay reagent composition; the exact procedure is dictated by the inter-reactivity of the assay constituents and the order in which they may have to react with the analyte of interest.
In the case of the DLFH, the lateral flow invention it can utilize any form of absorbent, solid phase carrier that is capable of transporting a fluid. These can include filter paper, cellulose or synthetic resins. More specifically, the lateral flow material can include cellulose, cellulose acetate, nitrocellulose, mixed ester, teflon, polyvinylidene difluoride (PVDF), polytetrafluoroethylene (PTFE), polysulfone, cotton linter, non-woven rayon, glass fiber, nylon, ion exchange or other suitable membranes or solid support.
After impregnation, the dipsticks are dried, cut into strips, glued to a support structure (usually a flexible, flat, plastic stick made up of polystyrene, vinyl polypropylene, and polyester or other suitable support material) as part of a xe2x80x9csandwichxe2x80x9d composed of the handle, test pad, and a synthetic resin film and/or a fine-mesh material in the manner described in German Pat. No, 2,118,455. In addition, the instant invention may be combined with the water-stable film as taught in U.S. Pat. No. 3,530,957 to produce a dipstick in which the excess sample fluid can be wiped off in order to improve the accuracy and precision of the results.
The sensitivity of the assay can be as low as 5 ug/mL, is the cutoffs illustrated in the present art are merely illustrative. The cutoff of 50.0 ug/mL is used because this is the range that an analgesic (high) effect is felt by the user or victim.